Proteomic analysis optimization: selective protein sample on-column retention in reverse-phase liquid chromatography.
In an effort to optimize reverse-phase liquid chromatography (RPLC) for proteomics, we studied the impact of composition of the sample injection solution on protein on-column selection and retention. All the proteins studied were retained on-column when injections were made in 50% formic acid, 0.1% TFA or 8.3M urea. When formic acid was increased to 80%, the superoxide dismutase standard (MW 26,159) and 58 mouse microsomal proteins that possessed low-range molecular weights, high pIs or basic amino acid clusters were non-retained, resulting in retention selectivity during sample injection. Introducing to the 80% formic acid injection solution an organic solvent such as acetonitrile or acetonitrile-DMSO induced further retention selectivity, and increasing levels of organic solvents reduced on-column retention. The proteome was split into the proteins that were retained on-column which eluted at higher retention times (RTs), vs the proteins that collected in the injection flow-through which normally eluted at lower RTs. This protein selectivity was confirmed after fraction collection, 1D-GE and nano-LC-MS/MS. The significance of this procedure is that it can be exploited for fast extraction of small basic proteins from the bulk of the proteome and for on-column enrichment of hydrophobic proteins.
Winnik
Witold M
WM
National Health and Environmental Effects Research Laboratory, Environmental Carcinogenesis Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, United States. winnik.witold@epa.gov
Ortiz
Pedro A
PA
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D005561:Formates; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000737:chemistry; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012997:Solvents; D053719:Tandem Mass Spectrometry
D005561:Formates; D010455:Peptides; D011506:Proteins; D012997:Solvents; C030544:formic acid